TktA

ABSTRACT

The invention provides tktA polypeptides and polynucleotides encoding tktA polypeptides and methods for producing such polypeptides by recombinant techniques. Also provided are methods for utilizing ttA polypeptides to screen for antibacterial compounds.

FIELD OF THE INVENTION

[0001] This invention relates to newly identified polynucleotides andpolypeptides, and their production and uses, as well as their variants,agonists and antagonists, and their uses. In particular, the inventionrelates to polynucleotides and polypeptides of the transketolase family,as well as their variants, herein referred to as “tkA,” “tktApolynucleotide(s),” and “tktA polypeptide(s)” as the case may be.

BACKGROUND OF THE INVENTION

[0002] Pseudomonas spp. are organisms that are widespread and foundcommonly in water, soil and on plants. They are opportunistic pathogensand generally do not cause fatal diseases in healthy individuals.However, these organisms are responsible for considerable morbidity andmortality in patients whose normal defense mechanisms have beenbreached. Pseudomonads are of particular concern in hospitals because oftheir ability to survive in aqueous solutions such as disinfectants,irrigation fluids, and dialysis fluids. The species P. aeruginosa isimplicated frequently in cases of nosocomial infection, and it is themost important human pathogen of its genera. P. aeruginosa is extremelyadaptable and can survive in relatively hostile environments. Outsidethe hospital setting, it may be found in many moist environments,including sink traps, baths, hot tubs, swimming pools, cosmetics, andsneakers. The pathogenicity of this organism is attributed to the widerange of potent virulence factors that it produces, including proteases,exotoxins, endotoxins, and hemolysins. P. aeruginosa utilizes thesevirulence factors to cause a spectrum of diseases from superficial skininfections to acute bacterial sepsis. Diseases caused by P. aeruginosaare particularly difficult to treat because the organism is naturallyresistant to most antibiotics. It has also been shown to developadditional antibiotic resistances after cessation of therapy, anduntreatable strains are often isolated from patients with chronic lunginfections (i.e. cystic fibrosis). Currently, the only availabletherapies for P. aeruginosa infections include fluoroquinolones,amikacin, gentamicin, and some of the newer broad-spectrum B-lactamantibiotics.

[0003] The frequency of Pseudomonas aeruginosa infections has risendramatically in the past few decades. This has been attributed to theemergence of multiply antibiotic resistant strains and an increasingpopulation of people with weakened immune systems. It is no longeruncommon to isolate Pseudomonas aeruginosa strains that are resistant tosome or all of the standard antibiotics. This phenomenon has created anunmet medical need and demand for new anti-microbial agents, vaccines,drug screening methods, and diagnostic tests for this organism.

[0004] Moreover, the drug discovery process is currently undergoing afundamental revolution as it embraces “functional genomics,” that is,high throughput genome- or gene-based biology. This approach is rapidlysuperseding earlier approaches based on “positional cloning” and othermethods. Functional genomics relies heavily on the various tools ofbioinformatics to identify gene sequences of potential interest from themany molecular biology databases now available as well as from othersources. There is a continuing and significant need to identify andcharacterize further genes and other polynucleotides sequences and theirrelated polypeptides, as targets for drug discovery.

[0005] Clearly, there exists a need for polynucleotides andpolypeptides, such as the tktA embodiments of the invention, that have apresent benefit of, among other things, being useful to screen compoundsfor antimicrobial activity. Such factors are also useful to determinetheir role in pathogenesis of infection, dysfunction and disease. Thereis also a need for identification and characterization of such factorsand their antagonists and agonists to find ways to prevent, ameliorateor correct such infection, dysfunction and disease.

SUMMARY OF THE INVENTION

[0006] The present invention relates to tktA, in particular tktApolypeptides and tktA polynucleotides, recombinant materials and methodsfor their production. In another aspect, the invention relates tomethods for using such polypeptides and polynucleotides, includingtreatment of microbial diseases, amongst others. In a further aspect,the invention relates to methods for identifying agonists andantagonists using the materials provided by the invention, and fortreating microbial infections and conditions associated with suchinfections with the identified agonist or antagonist compounds. In astill further aspect, the invention relates to diagnostic assays fordetecting diseases associated with microbial infections and conditionsassociated with such infections, such as assays for detecting tktAexpression or activity.

[0007] Various changes and modifications within the spirit and scope ofthe disclosed invention will become readily apparent to those skilled inthe art from reading the following descriptions and from reading theother parts of the present disclosure.

DESCRIPTION OF THE INVENTION

[0008] The invention relates to tktA polypeptides and polynucleotides asdescribed in greater detail below. In particular, the invention relatesto polypeptides and polynucleotides of a tktA of Pseudomonas aeruginosa,that is related by amino acid sequence homology to E. coli tkt1polypeptide. The invention relates especially to tktA having anucleotide and amino acid sequences set out in Table 1 as SEQ ID NO:1and SEQ ID NO:2 respectively. Note that sequences recited in theSequence Listing below as “DNA” represent an exemplification of theinvention, since those of ordinary skill will recognize that suchsequences can be usefully employed in polynucleotides in general,including ribopolynucleotides. TABLE 1 tktA Polynucleotide andPolypeptide Sequences (A) Pseudomonas aeruginosa tktA polynucleotidesequence [SEQ ID NO:1].5′-ATGCCCAGCCGTCGTGAGCGAGCCAATGCCATCCGTGCACTGAGCATGGATGCCGTGCAGAAAGCCAACAGCGGCCACCCGGGCGCCCCGATGGGCATGGCCGATATCGCCGAGGTCCTCTGGCGCGACTACATGCAGCACAACCCGAGCAACCCGCAGTGGGCCAACCGCGACCGCTTCGTGCTGTCCAACGGCCACGGCTCGATGCTGATCTACTCCCTGCTGCACCTCACCGGGTACGACCTCGGCATCGAGGACCTGAAGAACTTCCGCCAGCTCAACTCGCGCACCCCGGGCCACCCGGAGTACGGCTACACCGCCGGCGTCGAGACCACCACCGGTCCGCTCGGCCAGGGCATCGCCAATGCGGTGGGCATGGCGCTGGCGGAGAAGGTCCTGGCCGCCCAGTTCAACCGCGACGGCCACGCGGTGGTCGACCACTACACCTACGCCTTCCTCGGCGACGGCTGCATGATGGAAGGCATTTCCCATGAGGTCGCCTCGCTGGCCGGCACCCTGCGCCTGAACAAGCTGATCGCCTTCTACGACGACAACGGCATTTCCATCGACGGCGAGGTCCACGGCTGGTTCACCGACGACACCCCGAAGCGCTTCGAGGCCTATGGCTGGCAAGTGATCCGCAACGTCGACGGGCATGACGCCGACGAGATCAAGACCGCCATCGATACCGCGCGCAAGAGCGACCAGCCGACCCTGATCTGCTGCAAGACCGTGATCGGTTTCGGCTCGCCGAACAAGCAGGGCAAGGAAGAGTGCCACGGCGCGCCGCTGGGCGCCGACGAGATCGCCGCGACCCGCGCCGCGCTGGGCTGGGAGCACGCTCCGTTCGAGATCCCGGCGCAGATCTACGCCGAGTGGGACGCCAAGGAAACCGGCGCCGCCCAGGAAGCCGAGTGGAACAAGCGTTTCGCCGCCTACCAGGCTGCCCATCCGGAACTGGCCGCCGAATTGCTGCGCCGCCTGAAGGGCGAGCTGCCGGCCGACTTCGCCGAGAAGGCCGCGGCCTACGTCGCCGATGTTGCCAACAAGGGTGAGACCATCGCCAGCCGCAAGGCCAGCCAGAACGCGCTGAACGCCTTCGGCCCGCTGCTGCCGGAGCTGCTCGGCGGTTCCGCCGACCTGGCCGGCTCCAACCTGACCTTGTGGAAGGGCTGCAAGGGCGTCAGCGCCGACGACGCCGCCGGCAACTACGTGTTCTACGGCGTGCGCGAATTCGGCATGAGCGCGATCATGAATGGCGTCGCCCTGCACGGCGGTTTCATTCCCTACGGTGCGACCTTCCTGATCTTCATGGAATACGCGCGCAACGCCGTGCGCATGTCCGCACTGATGAAGCAGCGCGTGCTCTACGTGTTCACCCACGACTCCATCGGCCTCGGCGAGGACGGCCCGACCCACCAGCCGATCGAACAACTGGCCAGCCTGCGCCTGACCCCGAACCTGGACACCTGGCGCCCGGCCGACGCGGTCGAGTCGGCGGTGGCCTGGAAGCATGCCATCGAGCGCGCCGACGGTCCGTCCGCGCTGATCTTCTCCCGCCAGAACCTGCCGCACCAGGCGCGCGACGTCGCCCAGGTGGCCGACATCGCCCGCGGCGGCTACGTGCTGAAGGACTGCGAAGGCGAGCCGGAACTGATCCTGATCGCCACCGGTTCGGAAGTCGGCCTGGCCGTGCAGGCCTACGACAAGCTCAGCGAGCAGGGCCGCAAGGTCCGCGTGGTATCGATGCCATGCACCAGCGTCTACGAGCAGCAGGACGAGTCCTACAAGCAGTCCGTGCTGCCGGTGGAAGTCGGCGCGCGCATCGCCATCGAGGCCGCCCATGCCGACTACTGGTACAAGTACGTCGGTCTCGACGGGCGCATCATCGGCATGACCAGCTTCGGCGAGTCGGCGCCGGCCCCGGCGCTGTTCGAGCACTTCGGCTTCACCCTGGACAACGTCCTGGCGGTGGGCGAGGAGCTGCTGGAAGACTGA-3′ (B) Pseudomonas aeruginosa tktA polypeptidesequence deduced from a polynucleotide sequence in this table [SEQ IDNO:2]. NH₂-MPSRRERANAIRALSMDAVQKANSGHPGAPMGMADIAEVLWRDYMQHNPSNPQWANRDRFVLSNGHGSMLIYSLLHLTGYDLGIEDLKNFRQLNSRTPGHPEYGYTAGVETTTGPLGQGIANAVGMALAEKVLAAQFNRDGHAVVDHYTYAFLGDGCMMEGISHEVASLAGTLRLHKLIAFYDDNGISIDGEVHGWFTDDTPKRFEAYGWQVIPNVDGHDADEIKTAIDTARKSDQPTLICCKTVIGFGSPNKQGKEECHGAPLGADEIAATRAALGWEHAPFEIPAQIYAEWDAKETGAAQEAEWNKRFAAYQAAHPELAAELLRRLKGELPADFAEKAAAYVADVANKGETIASRKASQNALNAFGPLLPELLGGSADLAGSNLTLWKGCKGVSADDAAGNYVFYGVREFGMSAIMNGVALHGGFIFYGATFLIFMEYAPNAVPMSALMKQRVLYVFTHDSIGLGEDGPTHQPIEQLASLRLTPNLDTWRPDAVESAVAWKHAIERADGPSAIJIFSRQNLPHQARDVAQVADIARGGYVLKDCEGEPELILIATGSEVGLAVQAYDKLSEQGRKVRVVSMPCTSVYEQQDESYKQSVLPVEVGARIAIFAAHADYWYKYVGLDGRIIGMTSFGESAPAPALFEHFGFTLDNVLAVGE ELLED-COOH

Polypeptides

[0009] TktA polypeptide of the invention is substantiallyphylogenetically related to other proteins of the transketolase family.

[0010] In one aspect of the invention there are provided polypeptides ofPseudomonas aeruginosa referred to herein as “tktA” and “tktApolypeptides” as well as biologically, diagnostically, prophylactically,clinically or therapeutically useful variants thereof, and compositionscomprising the same.

[0011] Among the particularly preferred embodiments of the invention arevariants of tktA polypeptide encoded by naturally occurring alleles of atktA gene.

[0012] The present invention further provides for an isolatedpolypeptide that: (a) comprises or consists of an amino acid sequencethat has at least 95% identity, most preferably at least 97-99% or exactidentity, to that of SEQ ID NO:2 over the entire length of SEQ ID NO:2;(b) a polypeptide encoded by an isolated polynucleotide comprising orconsisting of a polynucleotide sequence that has at least 95% identity,even more preferably at least 97-99% or exact identity to SEQ ID NO:1over the entire length of SEQ ID NO:1; (c) a polypeptide encoded by anisolated polynucleotide comprising or consisting of a polynucleotidesequence encoding a polypeptide that has at least 95% identity, evenmore preferably at least 97-99% or exact identity, to the amino acidsequence of SEQ ID NO:2, over the entire length of SEQ ID NO:2.

[0013] The polypeptides of the invention include a polypeptide of Table1 [SEQ ID NO:2] (in particular a mature polypeptide) as well aspolypeptides and fragments, particularly those that has a biologicalactivity of tktA, and also those that have at least 95% identity to apolypeptide of Table 1 [SEQ ID NO:2] and also include portions of suchpolypeptides with such portion of the polypeptide generally comprisingat least 30 amino acids and more preferably at least 50 amino acids.

[0014] The invention also includes a polypeptide consisting of orcomprising a polypeptide of the formula:

X—(R₁)_(m)—(R₂)—(R₃)_(n)—Y

[0015] wherein, at the amino terminus, X is hydrogen, a metal or anyother moiety described herein for modified polypeptides, and at thecarboxyl terminus, Y is hydrogen, a metal or any other moiety describedherein for modified polypeptides, R₁ and R₃ are any amino acid residueor modified amino acid residue, m is an integer between 1 and 1000 orzero, n is an integer between 1 and 1000 or zero, and R₂ is an aminoacid sequence of the invention, particularly an amino acid sequenceselected from Table 1 or modified forms thereof. In the formula above,R₂ is oriented so that its amino terminal amino acid residue is at theleft, covalently bound to R₁, and its carboxy terminal amino acidresidue is at the right, covalently bound to R₃. Any stretch of aminoacid residues denoted by either R₁ or R₃, where m and/or n is greaterthan 1, may be either a heteropolymer or a homopolymer, preferably aheteropolymer. Other preferred embodiments of the invention are providedwhere m is an integer between 1 and 50, 100 or 500, and n is an integerbetween 1 and 50, 100, or 500.

[0016] It is most preferred that a polypeptide of the invention isderived from Pseudomonas aeruginosa, however, it may preferably beobtained from other organisms of the same taxonomic genus. A polypeptideof the invention may also be obtained, for example, from organisms ofthe same taxonomic family or order.

[0017] A fragment is a variant polypeptide having an amino acid sequencethat is entirely the same as part but not all of any amino acid sequenceof any polypeptide of the invention. As with tktA polypeptides,fragments may be “free-standing,” or comprised within a largerpolypeptide of which they form a part or region, most preferably as asingle continuous region in a single larger polypeptide.

[0018] Preferred fragments include, for example, truncation polypeptideshaving a portion of an amino acid sequence of Table 1 [SEQ ID NO:2], orof variants thereof, such as a continuous series of residues thatincludes an amino- and/or carboxyl-terminal amino acid sequence.Degradation forms of the polypeptides of the invention produced by or ina host cell, particularly a Pseudomonas aeruginosa, are also preferred.Further preferred are fragments characterized by structural orfunctional attributes such as fragments that comprise alpha-helix andalpha-helix forming regions, beta-sheet and beta-sheet-forming regions,turn and turn-forming regions, coil and coil-forming regions,hydrophilic regions, hydrophobic regions, alpha amphipathic regions,beta amphipathic regions, flexible regions, surface-forming regions,substrate binding region, and high antigenic index regions.

[0019] Further preferred fragments include an isolated polypeptidecomprising an amino acid sequence having at least 15, 20, 30, 40, 50 or100 contiguous amino acids from the amino acid sequence of SEQ ID NO:2,or an isolated polypeptide comprising an amino acid sequence having atleast 15, 20, 30, 40, 50 or 100 contiguous amino acids truncated ordeleted from the amino acid sequence of SEQ ID NO:2.

[0020] Fragments of the polypeptides of the invention may be employedfor producing the corresponding full-length polypeptide by peptidesynthesis; therefore, these variants may be employed as intermediatesfor producing the full-length polypeptides of the invention.

Polynucleotides

[0021] It is an object of the invention to provide polynucleotides thatencode tktA polypeptides, particularly polynucleotides that encode apolypeptide herein designated tktA.

[0022] In a particularly preferred embodiment of the invention thepolynucleotide comprises a region encoding tktA polypeptides comprisinga sequence set out in Table 1 [SEQ ID NO:1] that includes a full lengthgene, or a variant thereof This invention provides that this fill lengthgene is essential to the growth and/or survival of an organism thatpossesses it, such as Pseudomonas aeruginosa.

[0023] As a further aspect of the invention there are provided isolatednucleic acid molecules encoding and/or expressing tktA polypeptides andpolynucleotides, particularly Pseudomonas aeruginosa tktA polypeptidesand polynucleotides, including, for example, unprocessed RNAs, ribozymeRNAs, mRNAs, cDNAs, genomic DNAs, B- and Z-DNAs. Further embodiments ofthe invention include biologically, diagnostically, prophylactically,clinically or therapeutically usefull polynucleotides and polypeptides,and variants thereof, and compositions comprising the same.

[0024] Another aspect of the invention relates to isolatedpolynucleotides, including at least one full length gene, that encodes atktA polypeptide having a deduced amino acid sequence of Table 1 [SEQ IDNO:2] and polynucleotides closely related thereto and variants thereof.

[0025] In another particularly preferred embodiment of the inventionthere is a tktA polypeptide from Pseudomonas aeruginosa comprising orconsisting of an amino acid sequence of Table 1 [SEQ ID NO:2], or avariant thereof.

[0026] Using the information provided herein, such as a polynucleotidesequence set out in Table 1 [SEQ ID NO:1], a polynucleotide of theinvention encoding tktA polypeptide may be obtained using standardcloning and screening methods, such as those for cloning and sequencingchromosomal DNA fragments from bacteria using Pseudomonas aeruginosa P.aeruginosa strain 4 cells as starting material, followed by obtaining afull length clone. For example, to obtain a polynucleotide sequence ofthe invention, such as a polynucleotide sequence given in Table 1 [SEQID NO:1], typically a library of clones of chromosomal DNA ofPseudomonas aeruginosa P. aeruginosa strain 4 in E.coli or some othersuitable host is probed with a radiolabeled oligonucleotide, preferablya 17-mer or longer, derived from a partial sequence. Clones carrying DNAidentical to that of the probe can then be distinguished using stringenthybridization conditions. By sequencing the individual clones thusidentified by hybridization with sequencing primers designed from theoriginal polypeptide or polynucleotide sequence it is then possible toextend the polynucleotide sequence in both directions to determine afull length gene sequence. Conveniently, such sequencing is performed,for example, using denatured double stranded DNA prepared from a plasmidclone. Suitable techniques are described by Maniatis, T., Fritsch, E. F.and Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, 2nd Ed.;Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y. (1989).(see in particular Screening By Hybridization 1.90 and SequencingDenatured Double-Stranded DNA Templates 13.70). Direct genomic DNAsequencing may also be performed to obtain a full length gene sequence.Illustrative of the invention, each polynucleotide set out in Table 1[SEQ ID NO:1] was discovered in a DNA library derived from Pseudomonasaeruginosa P. aeruginosa strain 4.

[0027] Moreover, each DNA sequence set out in Table 1 [SEQ ID NO:1]contains an open reading frame encoding a protein having about thenumber of amino acid residues set forth in Table 1 [SEQ ID NO:2] with adeduced molecular weight that can be calculated using amino acid residuemolecular weight values well known to those skilled in the art. Thepolynucleotide of SEQ ID NO:1, between nucleotide number 1 and the stopcodon that begins at nucleotide number 1996 of SEQ ID NO:1, encodes thepolypeptide of SEQ ID NO:2.

[0028] In a further aspect, the present invention provides for anisolated polynucleotide comprising or consisting of: (a) apolynucleotide sequence that has at least 95% identity, even morepreferably at least 97-99% or exact identity to SEQ ID NO:1 over theentire length of SEQ ID NO:1, or the entire length of that portion ofSEQ ID NO:1 which encodes SEQ ID NO:2; (b) a polynucleotide sequenceencoding a polypeptide that has at least 95% identity, even morepreferably at least 97-99% or 100% exact, to the amino acid sequence ofSEQ ID NO:2, over the entire length of SEQ ID NO:2.

[0029] A polynucleotide encoding a polypeptide of the present invention,including homologs and orthologs from species other than Pseudomonasaeruginosa, may be obtained by a process that comprises the steps ofscreening an appropriate library under stringent hybridizationconditions with a labeled or detectable probe consisting of orcomprising the sequence of SEQ ID NO:1 or a fragment thereof; andisolating a full-length gene and/or genomic clones comprising saidpolynucleotide sequence.

[0030] The invention provides a polynucleotide sequence identical overits entire length to a coding sequence (open reading frame) in Table 1[SEQ ID NO:1]. Also provided by the invention is a coding sequence for amature polypeptide or a fragment thereof, by itself as well as a codingsequence for a mature polypeptide or a fragment in reading frame withanother coding sequence, such as a sequence encoding a leader orsecretory sequence, a pre-, or pro- or prepro-protein sequence. Thepolynucleotide of the invention may also comprise at least onenon-coding sequence, including for example, but not limited to at leastone non-coding 5′ and 3′ sequence, such as the transcribed butnon-translated sequences, termination signals (such as rho-dependent andrho-independent termination signals), ribosome binding sites, Kozaksequences, sequences that stabilize mRNA, introns, and polyadenylationsignals. The polynucleotide sequence may also comprise additional codingsequence encoding additional amino acids. For example, a marker sequencethat facilitates purification of a fused polypeptide can be encoded. Incertain embodiments of the invention, the marker sequence is ahexa-histidine peptide, as provided in the pQE vector (Qiagen, Inc.) anddescribed in Gentz et al., Proc. Natl. Acad. Sci., USA 86: 821-824(1989), or an HA peptide tag (Wilson et al., Cell 37: 767 (1984), bothof that may be useful in purifing polypeptide sequence fused to them.Polynucleotides of the invention also include, but are not limited to,polynucleotides comprising a structural gene and its naturallyassociated sequences that control gene expression.

[0031] A preferred embodiment of the invention is a polynucleotide ofconsisting of or comprising nucleotide 1 to the nucleotide immediatelyupstream of or including nucleotide 1996 set forth in SEQ ID NO:1 ofTable 1, both of that encode a tktA polypeptide.

[0032] The invention also includes a polynucleotide consisting of orcomprising a polynucleotide of the formula:

X—(R₁)_(m)—(R₂)—(R₃)_(n)—Y

[0033] wherein, at the 5′ end of the molecule, X is hydrogen, a metal ora modified nucleotide residue, or together with Y defines a covalentbond, and at the 3′ end of the molecule, Y is hydrogen, a metal, or amodified nucleotide residue, or together with X defines the covalentbond, each occurrence of R₁ and R₃ is independently any nucleic acidresidue or modified nucleic acid residue, m is an integer between 1 and3000 or zero, n is an integer between 1 and 3000 or zero, and R₂ is anucleic acid sequence or modified nucleic acid sequence of theinvention, particularly a nucleic acid sequence selected from Table 1 ora modified nucleic acid sequence thereof. In the polynucleotide formulaabove, R₂ is oriented so that its 5′ end nucleic acid residue is at theleft, bound to R₁, and its 3′ end nucleic acid residue is at the right,bound to R₃. Any stretch of nucleic acid residues denoted by either R₁and/or R₂, where m and/or n is greater than 1, may be either aheteropolymer or a homopolymer, preferably a heteropolymer. Where, in apreferred embodiment, X and Y together define a covalent bond, thepolynucleotide of the above formula is a closed, circularpolynucleotide, that can be a double-stranded polynucleotide wherein theformula shows a first strand to which the second strand iscomplementary. In another preferred embodiment m and/or n is an integerbetween 1 and 1000. Other preferred embodiments of the invention areprovided where m is an integer between 1 and 50, 100 or 500, and n is aninteger between 1 and 50, 100, or 500.

[0034] It is most preferred that a polynucleotide of the invention isderived from Pseudomonas aeruginosa, however, it may preferably beobtained from other organisms of the same taxonomic genus. Apolynucleotide of the invention may also be obtained, for example, fromorganisms of the same taxonomic family or order.

[0035] The term “polynucleotide encoding a polypeptide” as used hereinencompasses polynucleotides that include a sequence encoding apolypeptide of the invention, particularly a bacterial polypeptide andmore particularly a polypeptide of the Pseudomonas aeruginosa tktAhaving an amino acid sequence set out in Table 1 [SEQ ID NO:2]. The termalso encompasses polynucleotides that include a single continuous regionor discontinuous regions encoding the polypeptide (for example,polynucleotides interrupted by integrated phage, an integrated insertionsequence, an integrated vector sequence, an integrated transposonsequence, or due to RNA editing or genomic DNA reorganization) togetherwith additional regions, that also may comprise coding and/or non-codingsequences.

[0036] The invention further relates to variants of the polynucleotidesdescribed herein that encode variants of a polypeptide having a deducedamino acid sequence of Table 1 [SEQ ID NO:2]. Fragments ofpolynucleotides of the invention may be used, for example, to synthesizefull-length polynucleotides of the invention.

[0037] Further particularly preferred embodiments are polynucleotidesencoding tktA variants, that have the amino acid sequence of tktApolypeptide of Table 1 [SEQ ID NO:2] in which several, a few, 5 to 10, 1to 5, 1 to 3, 2, 1 or no amino acid residues are substituted, modified,deleted and/or added, in any combination. Especially preferred amongthese are silent substitutions, additions and deletions, that do notalter the properties and activities of tktA polypeptide.

[0038] Preferred isolated polynucleotide embodiments also includepolynucleotide fragments, such as a polynucleotide comprising a nuclicacid sequence having at least 15, 20, 30, 40, 50 or 100 contiguousnucleic acids from the polynucleotide sequence of SEQ ID NO:1, or anpolynucleotide comprising a nucleic acid sequence having at least 15,20, 30, 40, 50 or 100 contiguous nucleic acids truncated or deleted fromthe 5′ and/or 3′ end of the polynucleotide sequence of SEQ ID NO:1.

[0039] Further preferred embodiments of the invention arepolynucleotides that are at least 95% or 97% identical over their entirelength to a polynucleotide encoding tktA polypeptide having an aminoacid sequence set out in Table 1 [SEQ ID NO:2], and polynucleotides thatare complementary to such polynucleotides. Most highly preferred arepolynucleotides that comprise a region that is at least 95% areespecially preferred. Furthermore, those with at least 97% are highlypreferred among those with at least 95%, and among these those with atleast 98% and at least 99% are particularly highly preferred, with atleast 99% being the more preferred.

[0040] Preferred embodiments are polynucleotides encoding polypeptidesthat retain substantially the same biological function or activity as amature polypeptide encoded by a DNA of Table 1 [SEQ ID NO:1].

[0041] In accordance with certain preferred embodiments of thisinvention there are provided polynucleotides that hybridize,particularly under stringent conditions, to tktA polynucleotidesequences, such as those polynucleotides in Table 1.

[0042] The invention further relates to polynucleotides that hybridizeto the polynucleotide sequences provided herein. In this regard, theinvention especially relates to polynucleotides that hybridize understringent conditions to the polynucleotides described herein. A specificexample of stringent hybridization conditions is overnight incubation at42° C. in a solution comprising:50% formamide, 5×SSC (150 mM NaCl, 15 mMtrisodium citrate), 50 mM sodium phosphate (pH7.6), 5×Denhardt'ssolution, 10% dextran sulfate, and 20 micrograms/ml of denatured,sheared salmon sperm DNA, followed by washing the hybridization supportin 0.1×SSC at about 65° C. Hybridization and wash conditions are wellknown and exemplified in Sambrook, et al., Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989),particularly Chapter 11 therein. Solution hybridization may also be usedwith the polynucleotide sequences provided by the invention.

[0043] The invention also provides a polynucleotide consisting of orcomprising a polynucleotide sequence obtained by screening anappropriate library comprising a complete gene for a polynucleotidesequence set forth in SEQ ID NO:1 under stringent hybridizationconditions with a probe having the sequence of said polynucleotidesequence set forth in SEQ ID NO:1 or a fragment thereof, and isolatingsaid polynucleotide sequence. Fragments useful for obtaining such apolynucleotide include, for example, probes and primers fully describedelsewhere herein.

[0044] As discussed elsewhere herein regarding polynucleotide assays ofthe invention, for instance, the polynucleotides of the invention, maybe used as a hybridization probe for RNA, cDNA and genomic DNA toisolate full length cDNAs and genomic clones encoding tktA and toisolate cDNA and genomic clones of other genes that have a highidentity, particularly high sequence identity, to a tktA gene. Suchprobes generally will comprise at least 15 nucleotide residues or basepairs. Preferably, such probes will have at least 30 nucleotide residuesor base pairs and may have at least 50 nucleotide residues or basepairs. Particularly preferred probes will have at least 20 nucleotideresidues or base pairs and will have lee than 30 nucleotide residues orbase pairs.

[0045] A coding region of a tktA gene may be isolated by screening usinga DNA sequence provided in Table 1 [SEQ ID NO:1] to synthesize anoligonucleotide probe. A labeled oligonucleotide having a sequencecomplementary to that of a gene of the invention is then used to screena library of cDNA, genomic DNA or mRNA to determine which members of thelibrary the probe hybridizes to.

[0046] There are several methods available and well known to thoseskilled in the art to obtain full-length DNAs, or extend short DNAs, forexample those based on the method of Rapid Amplification of cDNA ends(RACE) (see, for example, Frohman, et al., PNAS USA 85: 8998-9002,1988). Recent modifications of the technique, exemplified by theMarathon™ technology (Clontech Laboratories Inc.) for example, havesignificantly simplified the search for longer cDNAs. In the Marathon™technology, cDNAs have been prepared from mRNA extracted from a chosentissue and an ‘adaptor’ sequence ligated onto each end. Nucleic acidamplification (PCR) is then carried out to amplify the “missing” 5′ endof the DNA using a combination of gene specific and adaptor specificoligonucleotide primers. The PCR reaction is then repeated using“nested” primers, that is, primers designed to anneal within theamplified product (typically an adaptor specific primer that annealsfurther 3′ in the adaptor sequence and a gene specific primer thatanneals further 5′ in the selected gene sequence). The products of thisreaction can then be analyzed by DNA sequencing and a full-length DNAconstructed either by joining the product directly to the existing DNAto give a complete sequence, or carrying out a separate full-length PCRusing the new sequence information for the design of the 5′ primer.

[0047] The polynucleotides and polypeptides of the invention may beemployed, for example, as research reagents and materials for discoveryof treatments of and diagnostics for diseases, particularly humandiseases, as further discussed herein relating to polynucleotide assays.

[0048] The polynucleotides of the invention that are oligonucleotidesderived from a sequence of Table 1 [SEQ ID NOS:1 or 2] may be used inthe processes herein as described, but preferably for PCR, to determinewhether or not the polynucleotides identified herein in whole or in partare transcribed in bacteria in infected tissue. It is recognized thatsuch sequences will also have utility in diagnosis of the stage ofinfection and type of infection the pathogen has attained.

[0049] The invention also provides polynucleotides that encode apolypeptide that is a mature protein plus additional amino orcarboxyl-terminal amino acids, or amino acids interior to a maturepolypeptide (when a mature form has more than one polypeptide chain, forinstance). Such sequences may play a role in processing of a proteinfrom precursor to a mature form, may allow protein transport, maylengthen or shorten protein half-life or may facilitate manipulation ofa protein for assay or production, among other things. As generally isthe case in vivo, the additional amino acids may be processed away froma mature protein by cellular enzymes.

[0050] For each and every polynucleotide of the invention there isprovided a polynucleotide complementary to it. It is preferred thatthese complementary polynucleotides are fully complementary to eachpolynucleotide with which they are complementary.

[0051] A precursor protein, having a mature form of the polypeptidefused to one or more prosequences may be an inactive form of thepolypeptide. When prosequences are removed such inactive precursorsgenerally are activated. Some or all of the prosequences may be removedbefore activation. Generally, such precursors are called proproteins.

[0052] As will be recognized, the entire polypeptide encoded by an openreading frame is often not required for activity. Accordingly, it hasbecome routine in molecular biology to map the boundaries of the primarystructure required for activity with N-terminal and C-terminal deletionexperiments. These experiments utilize exonuclease digestion orconvenient restriction sites to cleave coding nucleic acid sequence. Forexample, Promega (Madison, Wis.) sell an Erase-abase™ system that usesExonuclease m designed to facilitate analysis of the deletion products(protocol available at www.protnega.com). The digested endpoints can berepaired (eg., by ligation to synthetic linkers) to the extent necessaryto preserve an open reading frame. In this way, the nucleic acid of SEQID NO:1 readily provides contiguous fragments of SEQ ID NO:2 sufficientto provide an activity, such as an enzymatic, binding orantibody-inducing activity. Nucleic acid sequences encoding suchfragments of SEQ ID NO:2 and variants thereof as described herein arewithin the invention, as are polypeptides so encoded.

[0053] As is known in the art, portions of the N-terminal and/orC-terminal sequence of a protein can generally be removed withoutserious consequence to the function of the protein. The amount ofsequence that can be removed is often quite substantial. The nucleicacid cutting and deletion methods used for creating such deletionvariants are now quite routine. Accordingly, any contiguous fragment ofSEQ ID NO:2 which retains at least 20%, preferably at least 50%, of anactivity of the polypeptide encoded by the gene for SEQ ID NO:2 iswithin the invention, as are corresponding fragment which are 70%, 80%,90%, 95%,97%, 98% or 99% identical to such contiguous fragments. In oneembodiment, the contiguous fragment comprises at least 70% of the aminoacid residues of SEQ ID NO:2, preferably at least 80%, 90% or 95% of theresidues.

[0054] In sum, a polynucleotide of the invention may encode a matureprotein, a mature protein plus a leader sequence (that may be referredto as a preprotein), a precursor of a mature protein having one or moreprosequences that are not the leader sequences of a preprotein, or apreproprotein, that is a precursor to a proprotein, having a leadersequence and one or more prosequences, that generally are removed duringprocessing steps that produce active and mature forms of thepolypeptide.

Vectors, Host Cells, Expression Systems

[0055] The invention also relates to vectors that comprise apolynucleotide or polynucleotides of the invention, host cells that aregenetically engineered with vectors of the invention and the productionof polypeptides of the invention by recombinant techniques. Cell-freetranslation systems can also be employed to produce such proteins usingRNAs derived from the DNA constructs of the invention.

[0056] Recombinant polypeptides of the present invention may be preparedby processes well known in those skilled in the art from geneticallyengineered host cells comprising expression systems. Accordingly, in afurther aspect, the present invention relates to expression systems thatcomprise a polynucleotide or polynucleotides of the present invention,to host cells that are genetically engineered with such expressionsystems, and to the production of polypeptides of the invention byrecombinant techniques.

[0057] For recombinant production of the polypeptides of the invention,host cells can be genetically engineered to incorporate expressionsystems or portions thereof or polynucleotides of the invention.Introduction of a polynucleotide into the host cell can be effected bymethods described in many standard laboratory manuals, such as Davis, etal., BASIC METHODS IN MOLECULAR BIOLOGY, (1986) and Sambrook, et al.,MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989), such as, calciumphosphate transfection, DEAE-dextran mediated transfection,transvection, microinjection, cationic lipid-mediated transfection,electroporation, transduction, scrape loading, ballistic introductionand infection.

[0058] Representative examples of appropriate hosts include bacterialcells, such as cells of streptococci, staphylococci, enterococci E.coli, streptomyces, cyanobacteria, Bacillus subtilis, and Pseudomonasaeruginosa; fungal cells, such as cells of a yeast, Kluveromyces,Saccharomyces, a basidiomycete, Candida albicans and Aspergillus; insectcells such as cells of Drosophila S2 and Spodoptera Sf9; animal cellssuch as CHO, COS, HeLa, C127, 3T3, BHK, 293, CV-1 and Bowes melanomacells; and plant cells, such as cells of a gymnosperm or angiosperm.

[0059] A great variety of expression systems can be used to produce thepolypeptides of the invention. Such vectors include, among others,chromosomal-, episomal- and virus-derived vectors, for example, vectorsderived from bacterial plasmids, from bacteriophage, from transposons,from yeast episomes, from insertion elements, from yeast chromosomalelements, from viruses such as baculoviruses, papova viruses, such asSV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabiesviruses, picomaviruses and retroviruses, and vectors derived fromcombinations thereof, such as those derived from plasmid andbacteriophage genetic elements, such as cosmids and phagemids. Theexpression system constructs may comprise control regions that regulateas well as engender expression. Generally, any system or vector suitableto maintain, propagate or express polynucleotides and/or to express apolypeptide in a host may be used for expression in this regard. Theappropriate DNA sequence may be inserted into the expression system byany of a variety of well-known and routine techniques, such as, forexample, those set forth in Sambrook et al., MOLECULAR CLONING, ALABORATORY MANUAL, (supra).

[0060] In recombinant expression systems in eukaryotes, for secretion ofa translated protein into the lumen of the endoplasmic reticulum, intothe periplasmic space or into the extracellular environment, appropriatesecretion signals may be incorporated into the expressed polypeptide.These signals may be endogenous to the polypeptide or they may beheterologous signals.

[0061] Polypeptides of the invention can be recovered and purified fromrecombinant cell cultures by well-known methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography, and lectin chromatography. Most preferably, highperformance liquid chromatography is employed for purification. Wellknown techniques for refolding protein may be employed to regenerateactive conformation when the polypeptide is denatured during isolationand or purification.

[0062] Diagnostic, Prognostic, Serotyping and Mutation Assays

[0063] This invention is also related to the use of tktA polynucleotidesand polypeptides of the invention for use as diagnostic reagents.Detection of tktA polynucleotides and/or polypeptides in a eukaryote,particularly a mammal, and especially a huran, will provide a diagnosticmethod for diagnosis of disease, staging of disease or response of aninfectious organism to drugs. Eukaryotes, particularly mammals, andespecially humans, particularly those infected or suspected to beinfected with an organism comprising the tktA gene or protein, may bedetected at the nucleic acid or amino acid level by a variety of wellknown techniques as well as by methods provided herein.

[0064] Polypeptides and polynucleotides for prognosis, diagnosis orother analysis may be obtained from a putatively infected and/orinfected individual's bodily materials. Polynucleotides from any ofthese sources, particularly DNA or RNA, may be used directly fordetection or may be amplified enzymatically by using PCR or any otheramplification technique prior to analysis. RNA, particularly mRNA, cDNAand genomic DNA may also be used in the same ways. Using amplification,characterization of the species and strain of infectious or residentorganism present in an individual, may be made by an analysis of thegenotype of a selected polynucleotide of the organism. Deletions andinsertions can be detected by a change in size of the amplified productin comparison to a genotype of a reference sequence selected from arelated organism, preferably a different species of the same genus or adifferent strain of the same species. Point mutations can be identifiedby hybridizing amplified DNA to labeled tktA polynucleotide sequences.Perfectly or significantly matched sequences can be distinguished fromimperfectly or more significantly mismatched duplexes by DNase or RNasedigestion, for DNA or RNA respectively, or by detecting differences inmelting temperatures or renaturation kinetics. Polynucleotide sequencedifferences may also be detected by alterations in the electrophoreticmobility of polynucleotide fragments in gels as compared to a referencesequence. This may be carried out with or without denaturing agents.Polynucleotide differences may also be detected by direct DNA or RNAsequencing. See, for example, Myers et al., Science, 230: 1242 (1985).Sequence changes at specific locations also may be revealed by nucleaseprotection assays, such as RNase, V1 and S1 protection assay or achemical cleavage method. See, for example, Cotton et a., Proc. Natl.Acad. Sci., USA, 85: 4397-4401 (1985).

[0065] In another embodiment, an array of oligonucleotides probescomprising tktA nucleotide sequence or fragments thereof can beconstructed to conduct efficient screening of, for example, geneticmutations, serotype, taxonomic classification or identification. Arraytechnology methods are well known and have general applicability and canbe used to address a variety of questions in molecular geneticsincluding gene expression, genetic linkage, and genetic variability(see, for example, Chee et al., Science, 274: 610 (1996)).

[0066] Thus in another aspect, the present invention relates to adiagnostic kit that comprises: (a) a polynucleotide of the presentinvention, preferably the nucleotide sequence of SEQ ID NO:1, or afragment thereof; (b) a nucleotide sequence complementary to that of(a); (c) a polypeptide of the present invention, preferably thepolypeptide of SEQ ID NO:2 or a fragment thereof; or (d) an antibody toa polypeptide of the present invention, preferably to the polypeptide ofSEQ ID NO:2. It will be appreciated that in any such kit, (a), (b), (c)or (d) may comprise a substantial component. Such a kit will be of usein diagnosing a disease or susceptibility to a Disease, among others.

[0067] This invention also relates to the use of polynucleotides of thepresent invention as diagnostic reagents. Detection of a mutated form ofa polynucleotide of the invention, preferable, SEQ ID NO:1, that isassociated with a disease or pathogenicity will provide a diagnostictool that can add to, or define, a diagnosis of a disease, a prognosisof a course of disease, a determination of a stage of disease, or asusceptibility to a disease, that results from under-expression,over-expression or altered expression of the polynucleotide. Organisms,particularly infectious organisms, carrying mutations in suchpolynucleotide may be detected at the polynucleotide level by a varietyof techniques, such as those described elsewhere herein.

[0068] The differences in a polynucleotide and/or polypeptide sequencebetween organisms possessing a first phenotype and organisms possessinga different, second different phenotype can also be determined. If amutation is observed in some or all organisms possessing the firstphenotype but not in any organisms possessing the second phenotype, thenthe mutation is likely to be the causative agent of the first phenotype.

[0069] Cells from an organism caring mutations or polymorphisms (allelicvariations) in a polynucleotide and/or polypeptide of the invention mayalso be detected at the polynucleotide or polypeptide level by a varietyof techniques, to allow for serotyping, for example. For example, RT-PCRcan be used to detect mutations in the RNA. It is particularly preferredto use RT-PCR in conjunction with automated detection systems, such as,for example, GeneScan. RNA, cDNA or genomic DNA may also be used for thesame purpose, PCR. As an example, PCR primers complementary to apolynucleotide encoding tktA polypeptide can be used to identify andanalyze mutations. The invention further provides these primers with 1,2, 3 or 4 nucleotides removed from the 5′ and/or the 3′ end. Theseprimers may be used for, among other things, amplifying tktA DNA and/orRNA isolated from a sample derived from an individual, such as a bodilymaterial. The primers may be used to amplify a polynucleotide isolatedfrom an infected individual, such that the polynucleotide may then besubject to various techniques for elucidation of the polynucleotidesequence. In this way, mutations in the polynucleotide sequence may bedetected and used to diagnose and/or prognose the infection or its stageor course, or to serotype and/or classify the infectious agent.

[0070] The invention further provides a process for diagnosing, disease,preferably bacterial infections, more preferably infections caused byPseudomonas aeruginosa, comprising determining from a sample derivedfrom an individual, such as a bodily material, an increased level ofexpression of polynucleotide having a sequence of Table 1 [SEQ ID NO:1].Increased or decreased expression of a tktA polynucleotide can bemeasured using any on of the methods well known in the art for thequantitation of polynucleotides, such as, for example, amplification,PCR, RT-PCR, RNase protection, Northern blotting, spectrometry and otherhybridization methods.

[0071] In addition, a diagnostic assay in accordance with the inventionfor detecting over-expression of tktA polypeptide compared to normalcontrol tissue samples may be used to detect the presence of aninfection, for example. Assay techniques that can be used to determinelevels of a tktA polypeptide, in a sample derived from a host, such as abodily material, are well-known to those of skill in the art. Such assaymethods include radioimmunoassays, competitive-binding assays, WesternBlot analysis, antibody sandwich assays, antibody detection and ELISAassays.

Antagonists and Agonists—Assays and Molecules

[0072] Polypeptides and polynucleotides of the invention may also beused to assess the binding of small molecule substrates and ligands in,for example, cells, cell-free preparations, chemical libraries, andnatural product mixtures. These substrates and ligands may be naturalsubstrates and ligands or may be structural or functional mimetics. See,e.g., Coligan et al., Current Protocols in Immunology 1(2): Chapter 5(1991).

[0073] Polypeptides and polynucleotides of the present invention areresponsible for many biological functions, including many diseasestates, in particular the Diseases herein mentioned. It is thereforedesirable to devise screening methods to identify compounds that agonize(e.g., stimulate) or that antagonize (e.g.,inhibit) the function of thepolypeptide or polynucleotide. Accordingly, in a further aspect, thepresent invention provides for a method of screening compounds toidentify those that agonize or that antagonize the function of apolypeptide or polynucleotide of the invention, as well as relatedpolypeptides and polynucleotides. In general, agonists or antagonists(e.g., inhibitors) may be employed for therapeutic and prophylacticpurposes for such Diseases as herein mentioned. Compounds may beidentified from a variety of sources, for example, cells, cell-freepreparations, chemical libraries, and natural product mixtures. Suchagonists and antagonists so-identified may be natural or modifiedsubstrates, ligands, receptors, enzymes, etc., as the case may be, oftktA polypeptides and polynucleotides; or may be structural orfunctional mimetics thereof (see Coligan et al., Current Protocols inImmunology 1(2):Chapter 5 (1991)).

[0074] The screening methods may simply measure the binding of acandidate compound to the polypeptide or polynucleotide, or to cells ormembranes bearing the polypeptide or polynucleotide, or a fusion proteinof the polypeptide by means of a label directly or indirectly associatedwith the candidate compound. Alternatively, the screening method mayinvolve competition with a labeled competitor. Further, these screeningmethods may test whether the candidate compound results in a signalgenerated by activation or inhibition of the polypeptide orpolynucleotide, using detection systems appropriate to the cellscomprising the polypeptide or polynucleotide. Inhibitors of activationare generally assayed in the presence of a known agonist and the effecton activation by the agonist by the presence of the candidate compoundis observed. Constitutively active polypeptide and/or constitutivelyexpressed polypeptides and polynucleotides may be employed in screeningmethods for inverse agonists, in the absence of an agonist orantagonist, by testing whether the candidate compound results ininhibition of activation of the polypeptide or polynucleotide, as thecase may be. Further, the screening methods may simply comprise thesteps of mixing a candidate compound with a solution comprising apolypeptide or polynucleotide of the present invention, to form amixture, measuring tktA polypeptide and/or polynucleotide activity inthe mixture, and comparing the tktA polypeptide and/or polynucleotideactivity of the mixture to a standard. Fusion proteins, such as thosemade from Fc portion and tktA polypeptide, as herein described, can alsobe used for high-throughput screening assays to identify antagonists ofthe polypeptide of the present invention, as well as of phylogeneticallyand and/or functionally related polypeptides (see D. Bennett et al., JMol Recognition, 8:52-58 (1995); and K. Johanson et al., J Biol Chem,270(16):9459-9471 (1995)).

[0075] The polynucleotides, polypeptides and antibodies that bind toand/or interact with a polypeptide of the present invention may also beused to configure screening methods for detecting the effect of addedcompounds on the production of mRNA and/or polypeptide in cells. Forexample, an ELISA assay may be constructed for measuring secreted orcell associated levels of polypeptide using monoclonal and polyclonalantibodies by standard methods known in the art. This can be used todiscover agents that may inhibit or enhance the production ofpolypeptide (also called antagonist or agonist, respectively) fromsuitably manipulated cells or tissues.

[0076] The invention also provides a method of screening compounds toidentify those that enhance (agonist) or block (antagonist) the actionof tktA polypeptides or polynucleotides, particularly those compoundsthat are bacteristatic and/or bactericidal. The method of screening mayinvolve high-throughput techniques. For example, to screen for agonistsor antagonists, a synthetic reaction mix, a cellular compartment, suchas a membrane, cell envelope or cell wall, or a preparation of anythereof, comprising tktA polypeptide and a labeled substrate or ligandof such polypeptide is incubated in the absence or the presence of acandidate molecule that may be a tktA agonist or antagonist. The abilityof the candidate molecule to agonize or antagonize the tk-A polypeptideis reflected in decreased binding of the labeled ligand or decreasedproduction of product from such substrate. Molecules that bindgratuitously, i.e., without inducing the effects of tktA polypeptide aremost likely to be good antagonists. Molecules that bind well and, as thecase may be, increase the rate of product production from substrate,increase signal transduction, or increase chemical channel activity areagonists. Detection of the rate or level of, as the case may be,production of product from substrate, signal transducton, or chemicalchannel activity may be enhanced by using a reporter system. Reportersystems that may be useful in this regard include but are not limited tocolorimetric, labeled substrate converted into product, a reporter genethat is responsive to changes in tktA polynucleotide or polypeptideactivity, and binding assays known in the art.

[0077] Polypeptides of the invention may be used to identify membranebound or soluble receptors, if any, for such polypeptide, throughstandard receptor binding techniques known in the art. These techniquesinclude, but are not limited to, ligand binding and crosslinking assaysin which the polypeptide is labeled with a radioactive isotope (forinstance, ¹²⁵¹I), chemically modified (for instance, biotinylated), orfused to a peptide sequence suitable for detection or purification, andincubated with a source of the putative receptor (e.g., cells, cellmembranes, cell supernatants, tissue extracts, bodily materials). Othermethods include biophysical techniques such as surface plasmon resonanceand spectroscopy. These screening methods may also be used to identifyagonists and antagonists of the polypeptide that compete with thebinding of the polypeptide to its receptor(s), if any. Standard methodsfor conducting such assays are well understood in the art.

[0078] The fluorescence polarization value for a fluorescently-taggedmolecule depends on the rotational correlation time or tumbling rate.Protein complexes, such as formed by tktA polypeptide associating withanother tktA polypeptide or other polypeptide, labeled to comprise afluorescently-labeled molecule will have higher polarization values thana fluorescently labeled monomeric protein. It is preferred that thismethod be used to characterize small molecules that disrupt polypeptidecomplexes.

[0079] Fluorescence energy transfer may also be used characterize smallmolecules that interfere with the formation of tktA polypeptide dimers,trimers, tetramers or higher order structures, or structures formed bytktA polypeptide bound to another polypeptide. TktA polypeptide can belabeled with both a donor and acceptor fluorophore. Upon mixing of thetwo labeled species and excitation of the donor fluorophore,fluorescence energy transfer can be detected by observing fluorescenceof the acceptor. Compounds that block dimerization will inhibitfluorescence energy transfer.

[0080] Surface plasmon resonance can be used to monitor the effect ofsmall molecules on tktA polypeptide self-association as well as anassociation of tktA polypeptide and another polypeptide or smallmolecule. TktA polypeptide can be coupled to a sensor chip at low sitedensity such that covalently bound molecules will be monomeric. Solutionprotein can then passed over the tktA polypeptide -coated surface andspecific binding can be detected in real-time by monitoring the changein resonance angle caused by a change in local refractive index. Thistechnique can be used to characterize the effect of small molecules onkinetic rates and equilibrium binding constants for tktA polypeptideself-association as well as an association of tktA polypeptide andanother polypeptide or small molecule.

[0081] A scintillation proximity assay may be used to characterize theinteraction between an association of tktA polypeptide with another tktApolypeptide or a different polypeptide. TktA polypeptide can be coupledto a scintillation-filled bead. Addition of radio-labeled tktApolypeptide results in binding where the radioactive source molecule isin close proximity to the scintillation fluid. Thus, signal is emittedupon tktA polypeptide binding and compounds that prevent tktApolypeptide self-association or an association of tktA polypeptide andanother polypeptide or small molecule will diminish signal.

[0082] In other embodiments of the invention there are provided methodsfor identifying compounds that bind to or otherwise interact with andinhibit or activate an activity or expression of a polypeptide and/orpolynucleotide of the invention comprising: contacting a polypeptideand/or polynucleotide of the invention with a compound to be screenedunder conditions to permit binding to or other interaction between thecompound and the polypeptide and/or polynucleotide to assess the bindingto or other interaction with the compound, such binding or interactionpreferably being associated with a second component capable of providinga detectable signal in response to the binding or interaction of thepolypeptide and/or polynucleotide with the compound; and determiningwhether the compound binds to or otherwise interacts with and activatesor inhibits an activity or expression of the polypeptide and/orpolynucleotide by detecting the presence or absence of a signalgenerated from the binding or interaction of the compound with thepolypeptide and/or polynucleotide.

[0083] Another example of an assay for tktA agonists is a competitiveassay that combines tktA and a potential agonist with tktA-bindingmolecules, recombinant tktA binding molecules, natural substrates orligands, or substrate or ligand mimetics, under appropriate conditionsfor a competitive inhibition assay. TktA can be labeled, such as byradioactivity or a colorimetric compound, such that the number of tktAmolecules bound to a binding molecule or converted to product can bedetermined accurately to assess the effectiveness of the potentialantagonist.

[0084] It will be readily appreciated by the skilled artisan that apolypeptide and/or polynucleotide of the present invention may also beused in a method for the structure-based design of an agonist orantagonist of the polypeptide and/or polynucleotide, by: (a) determiningin the first instance the three-dimensional structure of the polypeptideand/or polynucleotide, or complexes thereof, (b) deducing thethree-dimensional structure for the likely reactive site(s), bindingsite(s) or motif(s) of an agonist or antagonist; (c) synthesizingcandidate compounds that are predicted to bind to or react with thededuced binding site(s), reactive site(s), and/or motif(s); and (d)testing whether the candidate compounds are indeed agonists orantagonists. It will be further appreciated that this will normally bean iterative process, and this iterative process may be performed usingautomated and computer-controlled steps.

[0085] In a further aspect, the present invention provides methods oftreating abnormal conditions such as, for instance, a Disease, relatedto either an excess of, an under-expression of, an elevated activity of,or a decreased activity of tktA polypeptide and/or polynucleotide.

[0086] If the expression and/or activity of the polypeptide and/orpolynucleotide is in excess, several approaches are available. Oneapproach comprises administering to an individual in need thereof aninhibitor compound (antagonist) as herein described, optionally incombination with a pharmaceutically acceptable carrier, in an amounteffective to inhibit the function and/or expression of the polypeptideand/or polynucleotide, such as, for example, by blocking the binding ofligands, substrates, receptors, enzymes, etc., or by inhibiting a secondsignal, and thereby alleviating the abnormal condition. In anotherapproach, soluble forms of the polypeptides still capable of binding theligand, substrate, enzymes, receptors, etc. in competition withendogenous polypeptide and/or polynucleotide may be administered.Typical examples of such competitors include fragments of the tktApolypeptide and/or polypeptide.

[0087] In still another approach, expression of the gene encodingendogenous tktA polypeptide can be inhibited using expression blockingtechniques. This blocking may be targeted against any step in geneexpression, but is preferably targeted against transcription and/ortranslation. An examples of a known technique of this sort involve theuse of antisense sequences, either internally generated or separatelyadministered (see, for example, O'Connor, J Neurochem (1991) 56:560 inOligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRCPress, Boca Raton, Fla. (1988)). Alternatively, oligonucleotides thatform triple helices with the gene can be supplied (see, for example, Leeet al, Nucleic Acids Res (1979) 6:3073; Cooney et al., Science (1988)241:456; Dervan et al., Science (1991) 251:1360). These oligomers can beadministered per se or the relevant oligomers can be expressed in vivo.

[0088] Each of the polynucleotide sequences provided herein may be usedin the discovery and development of antibacterial compounds. The encodedprotein, upon expression, can be used as a target for the screening ofantibacterial drugs. Additionally, the polynucleotide sequences encodingthe amino terminal regions of the encoded protein or Shine-Delgarno orother translation facilitating sequences of the respective mRNA can beused to construct antisense sequences to control the expression of thecoding sequence of interest.

[0089] The invention also provides the use of the polypeptide,polynucleotide, agonist or antagonist of the invention to interfere withthe initial physical interaction between a pathogen or pathogens and aeukaryotic, preferably mammalian, host responsible for sequelae ofinfection. In particular, the molecules of the invention may be used: inthe prevention of adhesion of bacteria, in particular gram positiveand/or gram negative bacteria, to eukaryotic, preferably mammalian,extracellular matrix proteins on in-dwelling devices or to extracellularmatrix proteins in wounds; to block bacterial adhesion betweeneukaryotic, preferably mammalian, extracellular matrix proteins andbacterial tktA proteins that mediate tissue damage and/or; to block thenormal progression of pathogenesis in infections initiated other than bythe implantation of in-dwelling devices or by other surgical techniques.

[0090] In accordance with yet another aspect of the invention, there areprovided tktA agonists and antagonists, preferably bacteristatic orbactericidal agonists and antagonists.

[0091] The antagonists and agonists of the invention may be employed,for instance, to prevent, inhibit and/or treat diseases.

[0092] Antagonists of the invention include, among others, small organicmolecules, peptides, polypeptides and antibodies that bind to apolynucleotide and/or polypeptide of the invention and thereby inhibitor extinguish its activity or expression. Antagonists also may be smallorganic molecules, a peptide, a polypeptide such as a closely relatedprotein or antibody that binds the same sites on a binding molecule,such as a binding molecule, without inducing tktA-induced activities,thereby preventing the action or expression of tktA polypeptides and/orpolynucleotides by excluding tktA polypeptides and/or polynucleotidesfrom binding.

[0093] Antagonists of the invention also include a small molecule thatbinds to and occupies the binding site of the polypeptide therebypreventing binding to cellular binding molecules, such that normalbiological activity is prevented. Examples of small molecules includebut are not limited to small organic molecules, peptides or peptide-likemolecules. Other antagonists include antisense molecules (see Okano, JNeurochem. 56: 560 (1991); OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORSOF GENE EXPRESSION, CRC Press, Boca Raton, Fla. (1988), for adescription of these molecules). Preferred antagonists include compoundsrelated to and variants of tktA.

[0094] Other examples of polypeptide antagonists include antibodies or,in some cases, oligonucleotides or proteins that are closely related tothe ligands, substrates, receptors, enzymes, etc., as the case may be,of the polypeptide, e.g., a fragment of the ligands, substrates,receptors, enzymes, etc.; or small molecules that bind to thepolypeptide of the present invention but do not elicit a response, sothat the activity of the polypeptide is prevented.

[0095] Small molecules of the invention preferably have a molecularweight below 2,000 daltons, more preferably between 300 and 1,000daltons, and most preferably between 400 and 700 daltons. It ispreferred that these small molecules are organic molecules.

[0096]Helicobacter pylori (herein “H. pylori”) bacteria infect thestomachs of over one-third of the world's population causing stomachcancer, ulcers, and gastritis (International Agency for Research onCancer (1994) Schistosomes, Liver Flukes and Helicobacter Pylori(International Agency for Research on Cancer, Lyon, France,http://www.uicc.ch/ecp/ecp2904.htm). Moreover, the International Agencyfor Research on Cancer recently recognized a cause-and-effectrelationship between H. pylori and gastric adenocarcinoma, classifyingthe bacterium as a Group I (definite) carcinogen. Preferredantimicrobial compounds of the invention (agonists and antagonists oftktA polypeptides and/or polynucleotides) found using screens providedby the invention, or known in the art, particularly narrow-spectrumantibiotics, should be useful in the treatment of H. pylori infection.Such treatment should decrease the advent of H. pylori-induced cancers,such as gastrointestinal carcinoma. Such treatment should also prevent,inhibit and/or cure gastric ulcers and gastritis.

[0097] All publications and references, including but not limited topatents and patent applications, cited in this specification are hereinincorporated by reference in their entirety as if each individualpublication or reference were specifically and individually indicated tobe incorporated by reference herein as being fully set forth. Any patentapplication to which this application claims priority is alsoincorporated by reference herein in its entirety in the manner describedabove for publications and references.

GLOSSARY

[0098] The following definitions are provided to facilitateunderstanding of certain terms used frequently herein.

[0099] “Bodily material(s) means any material derived from an individualor from an organism infecting, infesting or inhabiting an individual,including but not limited to, cells, tissues and waste, such as, bone,blood, serum, cerebrospinal fluid, semen, saliva, muscle, cartilage,organ tissue, skin, urine, stool or autopsy materials..

[0100] “Disease(s)” means any disease caused by or related to infectionby a bacteria, including, for example, there is a wide range ofdiseases, both community- and hospital-acquired, which can be caused byP. aeruginosa infection. The most severe and life-threatening infectionsare endocarditis, bacteremia, and pneumonia. Respiratory tractinfections with P. aeruginosa are particularly problematic for intubatedpatients and for individuals with cystic fibrosis. In the latter, thisorganism produces chronic lung infections which ultimately lead to anearly demise. In addition, P. aeruginosa is commonly implicated inosteomyelitis, infections of the eye, “swimmer's ear,” otitis media,malignant otitis externa (which can lead to meningitis), folliculitis,urinry tract infections, and wound infections. For patients recoveringfrom severe burn wounds, P. aeruginosa is an especially intractablepathogen..

[0101] “Host cell(s)” is a cell that has been introduced (e.g.,transformed or transfected) or is capable of introduction (e.g.,transformation or transfection) by an exogenous polynucleotide sequence.

[0102] “Identity,” as known in the art, is a relationship between two ormore polypeptide sequences or two or more polynucleotide sequences, asthe case may be, as determined by comparing the sequences. In the art,“identity” also means the degree of sequence relatedness betweenpolypeptide or polynucleotide sequences, as the case may be, asdetermined by the match between strings of such sequences. “Identity”can be readily calculated by known methods, including but not limited tothose described in (Computational Molecular Biology, Lesk, A. M., ed.,Oxford University Press, New York, 1988; Biocomputing: Informatics andGenome Projects, Smith, D. W., ed., Academic Press, New York, 1993;Computer Analysis of sequence Data, Part I, Griffin, A. M., and Griffin,H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; and SequenceAnalysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press,New York, 1991; and Carillo, H., and Lipman, D., SIAM J Applied Math.,48: 1073 (1988). Methods to determine identity are designed to give thelargest match between the sequences tested. Moreover, methods todetermine identity are codified in publicly available computer programs.Computer program methods to determine identity between two sequencesinclude, but are not limited to, the GCG program package (Devereux, J.,et al., Nucleic Acids Research 12(1):387 (1984)), BLASTP, BLASTN, andFASTA (Altschul, S. F. et al., J Molec. Biol. 215: 403-410 (1990). TheBLAST X program is publicly available from NCBI and other sources (BLASTManual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894;Altschul, S., et al., J Mol. Biol. 215: 403-410 (1990). The well knownSmith Waterman algorithm may also be used to determine identity.

[0103] Parameters for polypeptide sequence comparison include thefollowing: Algorithm:

[0104] Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970)

[0105] Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc.Natl. Acad. Sci. USA. 89:10915-10919 (1992)

[0106] Gap Penalty:12

[0107] Gap Length Penalty:4

[0108] A program useful with these parameters is publicly available asthe “gap” program from Genetics Computer Group, Madison Wis. Theaforementioned parameters are the default parameters for peptidecomparisons (along with no penalty for end gaps).

[0109] Parameters for polynucleotide comparison include the following:Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970)

[0110] Comparison matrix: matches=+10, mismatch=0

[0111] Gap Penalty:50

[0112] Gap Length Penalty:3

[0113] Available as: The “gap” program from Genetics Computer Group,Madison Wis. These are the default parameters for nucleic acidcomparisons.

[0114] A preferred meaning for “identity” for polynucleotides andpolypeptides, as the case may be, are provided in (1) and (2) below.

[0115] (1) Polynucleotide embodiments further include an isolatedpolynucleotide comprising a polynucleotide sequence having at least a95, 97 or 100% identity to the reference sequence of SEQ ID NO:1,wherein said polynucleotide sequence may be identical to the referencesequence of SEQ ID NO:1 or may include up to a certain integer number ofnucleotide alterations as compared to the reference sequence, whereinsaid alterations are selected from the group consisting of at least onenucleotide deletion, substitution, including transition andtransversion, or insertion, and wherein said alterations may occur atthe 5′ or 3′ terminal positions of the reference nucleotide sequence oranywhere between those terminal positions, interspersed eitherindividually among the nucleotides in the reference sequence or in oneor more contiguous groups within the reference sequence, and whereinsaid number of nucleotide alterations is determined by multiplying thetotal number of nucleotides in SEQ ID NO:1 by the integer defining thepercent identity divided by 100 and then subtracting that product fromsaid total number of nucleotides in SEQ ID NO:1, or:

n _(n) <x _(n)−(x _(n) ·Y),

[0116] wherein n_(n) is the number of nucleotide alterations, x_(n) isthe total number of nucleotides in SEQ ID NO:1, y is 0.95 for 95%, 0.97for 97% or 1.00 for 100%, and · is the symbol for the multiplicationoperator, and wherein any non-integer product of x_(n) and y is roundeddown to the nearest integer prior to subtracting it from x_(n).Alterations of a polynucleotide sequence encoding the polypeptide of SEQID NO:2 may create nonsense, missense or frameshift mutations in thiscoding sequence and thereby alter the polypeptide encoded by thepolynucleotide following such alterations.

[0117] (2) Polypeptide embodiments further include an isolatedpolypeptide comprising a polypeptide having at least a 95, 97 or 100%identity to a polypeptide reference sequence of SEQ ID NO:2, whereinsaid polypeptide sequence may be identical to the reference sequence ofSEQ ID NO:2 or may include up to a certain integer number of amino acidalterations as compared to the reference sequence, wherein saidalterations are selected from the group consisting of at least one aminoacid deletion, substitution, including conservative and non-conservativesubstitution, or insertion, and wherein said alterations may occur atthe amino- or carboxy-terminal positions of the reference polypeptidesequence or anywhere between those terminal positions, interspersedeither individually among the amino acids in the reference sequence orin one or more contiguous groups within the reference sequence, andwherein said number of amino acid alterations is determined bymultiplying the total number of amino acids in SEQ ID NO:2 by theinteger defining the percent identity divided by 100 and thensubtracting that product from said total number of amino acids in SEQ IDNO:2, or:

n _(a) ≦X _(a)−(x _(a) ·Y),

[0118] wherein n_(a) is the number of amino acid alterations, x_(a) isthe total number of amino acids in SEQ ID NO:2, y is 0.95 for 95%, 0.97for 97% or 1.00 for 100%, and · is the symbol for the multiplicationoperator, and wherein any non-integer product of x_(a) and y is roundeddown to the nearest integer prior to subtracting it from x_(a).

[0119] “Individual(s)” means a multicellular eukaryote, including, butnot limited to a metazoan, a mammal, an ovid, a bovid, a simian, aprimate, and a human.

[0120] “Isolated” means altered “by the hand of man” from its naturalstate, i.e., if it occurs in nature, it has been changed or removed fromits original environment, or both. For example, a polynucleotide or apolypeptide naturally present in a living organism is not “isolated,”but the same polynucleotide or polypeptide separated from the coexistingmaterials of its natural state is “isolated”, as the term is employedherein. Moreover, a polynucleotide or polypeptide that is introducedinto an organism by transformation, genetic manipulation or by any otherrecombinant method is “isolated” even if it is still present in saidorganism, which organism may be living or non-living.

[0121] “Organism(s)” means a (i) prokaryote, including but not limitedto, a member of the genus Streptococcus, Staphylococcus, Bordetella,Corynebacterium, Mycobacterium, Neisseria, Haemophilus, Actinomycetes,Streptomycetes, Nocardia, Enterobacter, Yersinia, Fancisella,Pasturella, Moraxella, Acinetobacter, Erysipelothrix, Branhamella,Actinobacillus, Streptobacillus, Listeria, Calymmatobacterium, Brucella,Bacillus, Clostridium, Treponema, Escherichia, Salmonella, Kleibsiella,Vibrio, Proteus, Erwinia, Borrelia, Leptospira, Spirillum,Campylobacter, Shigella, Legionella, Pseudomonas, Aeromonas, Rickettsia,Chlamydia, Borrelia and Mycoplasma, and flirter including, but notlimited to, a member of the species or group, Group A Streptococcus,Group B Streptococcus, Group C Streptococcus, Group D Streptococcus,Group G Streptococcus, Streptococcus pneumoniae, Streptococcus pyogenes,Streptococcus agalactiae, Streptococcus faecalis, Streptococcus faecium,Streptococcus durans, Neisseria gonorrheae, Neisseria meningitidis,Staphylococcus aureus, Staphylococcus epidermidis, Corynebacteriumdiptheriae, Gardnerella vaginalis, Mycobacterium tuberculosis,Mycobacterium bovis, Mycobacterium ulcerans, Mycobacterium leprae,Actinomyctes israelii, Listeria monocytogenes, Bordetella pertusis,Bordatella parapertusis, Bordetella bronchiseptica, Escherichia coli,Shigella dysenteriae, Haemophilus influenzae, Haemophilus aegyptius,Haemophilus parainfluenzae, Haemophilus ducreyi, Bordetella, Salmonellatyphi, Citrobacter freundii, Proteus mirabilis, Proteus vulgaris,Yersinia pestis, Kleibsiella pneumoniae, Serratia marcessens, Serratialiquefaciens, Vibrio cholera, Shigella dysenterii, Shigella flexneri,Pseudomonas aeruginosa, Franscisella tularensis, Brucella abortis,Bacillus anthracis, Bacillus cereus, Clostridium perfringens,Clostridium tetani, Clostridium botulinum, Treponema pallidum,Rickettsia rickettsii and Chlamydia trachomitis, (ii) an archaeon,including but not limited to Archaebacter, and (iii) a unicellular orfilamentous eukaryote, including but not limited to, a protozoan, afungus, a member of the genus Saccharomyces, Kluveromyces, or Candida,and a member of the species Saccharomyces ceriviseae, Kluveromyceslactis, or Candida albicans.

[0122] “Polynucleotide(s)” generally refers to any polyribonucleotide orpolydeoxyribonucleotide, that may be unmodified RNA or DNA or modifiedRNA or DNA. “Polynucleotide(s)” include, without limitation, single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions or single-, double- and triple-stranded regions,single- and double-stranded RNA, and RNA that is mixture of single- anddouble-stranded regions, hybrid molecules comprising DNA and RNA thatmay be single-stranded or, more typically, double-stranded, ortriple-stranded regions, or a mixture of single- and double-strandedregions. In addition, “polynucleotide” as used herein refers totriple-stranded regions comprising RNA or DNA or both RNA and DNA. Thestrands in such regions may be from the same molecule or from differentmolecules. The regions may include all of one or more of the molecules,but more typically involve only a region of some of the molecules. Oneof the molecules of a triple-helical region often is an oligonucleotide.As used herein, the term “polynucleotide(s)” also includes DNAs or RNAsas described above that comprise one or more modified bases. Thus, DNAsor RNAs with backbones modified for stability or for other reasons are“polynucleotide(s)” as that term is intended herein. Moreover, DNAs orRNAs comprising unusual bases, such as inosine, or modified bases, suchas tritylated bases, to name just two examples, are polynucleotides asthe term is used herein. It will be appreciated that a great variety ofmodifications have been made to DNA and RNA that serve many usefulpurposes known to those of skill in the art. The term“polynucleotide(s)” as it is employed herein embraces such chemically,enzymatically or metabolically modified forms of polynucleotides, aswell as the chemical forms of DNA and RNA characteristic of viruses andcells, including, for example, simple and complex cells.“Polynucleotide(s)” also embraces short polynucleotides often referredto as oligonucleotide(s).

[0123] “Polypeptide(s)” refers to any peptide or protein comprising twoor more amino acids joined to each other by peptide bonds or modifiedpeptide bonds. “Polypeptide(s)” refers to both short chains, commonlyreferred to as peptides, oligopeptides and oligomers and to longerchains generally referred to as proteins. Polypeptides may compriseamino acids other than the 20 gene encoded amino acids. “Polypeptide(s)”include those modified either by natural processes, such as processingand other post-translational modifications, but also by chemicalmodification techniques. Such modifications are well described in basictexts and in more detailed monographs, as well as in a voluminousresearch literature, and they are well known to those of skill in theart. It will be appreciated that the same type of modification may bepresent in the same or varying degree at several sites in a givenpolypeptide. Also, a given polypeptide may comprise many types ofmodifications. Modifications can occur anywhere in a polypeptide,including the peptide backbone, the amino acid side-chains, and theamino or carboxyl termini. Modifications include, for example,acetylation, acylation, ADP-ribosylation, amidation, covalent attachmentof flavin, covalent attachment of a heme moiety, covalent attachment ofa nucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent cross-links, formation of cysteine, formation ofpyroglutamate, formylation, gamma-carboxylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,proteolytic processing, phosphorylation, prenylation, racemization,glycosylation, lipid attachment, sulfation, gamma-carboxylation ofglutamic acid residues, hydroxylation and ADP-ribosylation,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins, such as arginylation, and ubiquitination. See, forinstance, PROTEINS—STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E.Creighton, W. H. Freeman and Company, New York (1993) and Wold, F.,Posttranslational Protein Modifications: Perspectives and Prospects,pgs. 1-12 in POSTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C.Johnson, Ed., Academic Press, New York (1983); Seifter et al., Meth.Enzymol 182:626-646 (1990) and Rattan et al., Protein Synthesis:Posttranslational Modifications and Aging, Ann. N.Y. Acad. Sci. 663:48-62 (1992). Polypeptides may be branched or cyclic, with or withoutbranching. Cyclic, branched and branched circular polypeptides mayresult from post-translational natural processes and may be made byentirely synthetic methods, as well.

[0124] “Recombinant expression system(s)” refers to expression systemsor portions thereof or polynucleotides of the invention introduced ortransformed into a host cell or host cell lysate for the production ofthe polynucleotides and polypeptides of the invention.

[0125] Varaint(s)” as the term is used herein, is a polynucleotide orpolypeptide that differs from a reference polynucleotide or polypeptiderespectively, but retains essential properties. A typical variant of apolynucleotide differs in nucleotide sequence from another, referencepolynucleotide. Changes in the nucleotide sequence of the variant may ormay not alter the amino acid sequence of a polypeptide encoded by thereference polynucleotide. Nucleotide changes may result in amino acidsubstitutions, additions, deletions, fusion proteins and truncations inthe polypeptide encoded by the reference sequence, as discussed below. Atypical variant of a polypeptide differs in amino acid sequence fromanother, reference polypeptide. Generally, differences are limited sothat the sequences of the reference polypeptide and the variant areclosely similar overall and, in many regions, identical. A variant andreference polypeptide may differ in amino acid sequence by one or moresubstitutions, additions, deletions in any combination. A substituted orinserted amino acid residue may or may not be one encoded by the geneticcode. The present invention also includes include variants of each ofthe polypeptides of the invention, that is polypeptides that vary fromthe referents by conservative amino acid substitutions, whereby aresidue is substituted by another with like characteristics. Typicalsuch substitutions are among Ala, Val, Leu and lie; among Ser and Thr;among the acidic residues Asp and Glu; among Asn and Gln; and among thebasic residues Lys and Arg; or aromatic residues Phe and Tyr.Particularly preferred are variants in which several, 5-10, 1-5, 1-3,1-2 or 1 amino acids are substituted, deleted, or added in anycombination. A variant of a polynucleotide or polypeptide may be anaturally occurring such as an allelic variant, or it may be a variantthat is not known to occur naturally. Non-naturally occurring variantsof polynucleotides and polypeptides may be made by mutagenesistechniques, by direct synthesis, and by other recombinant methods knownto skilled artisans.

EXAMPLES

[0126] The examples below are carried out using standard techniques,that are well known and routine to those of skill in the art, exceptwhere otherwise described in detail. The examples are illustrative, butdo not limit the invention.

Example 1 Strain selection, Library Production and Sequencing

[0127] The polynucleotide having a DNA sequence given in Table 1 [SEQ IDNO:1] was obtained from a library of clones of chromosomal DNA ofPseudomonas aeruginosa in E. coli. The sequencing data from two or moreclones comprising overlapping Pseudomonas aeruginosa DNAs was used toconstruct the contiguous DNA sequence in SEQ ID NO:1. Libraries may beprepared by routine methods, for example:

[0128] Methods 1 and 2 below.

[0129] Total cellular DNA is isolated from Pseudomonas aeruginosa P.aeruginosa strain 4 according to standard procedures andsize-fractionated by either of two methods.

Method 1

[0130] Total cellular DNA is mechanically sheared by passage through aneedle in order to size-fractionate according to standard procedures.DNA fragments of up to 11 kbp in size are rendered blunt by treatmentwith exonuclease and DNA polymerase, and EcoRI linkers added. Fragmentsare ligated into the vector Lambda ZapII that has been cut with EcoRI,the library packaged by standard procedures and E.coli infected with thepackaged library. The library is amplified by standard procedures.

Method 2

[0131] Total cellular DNA is partially hydrolyzed with a one or acombination of restriction enzymes appropriate to generate a series offragments for cloning into library vectors (e.g., RsaI, PalI, AluI,Bshl235I), and such fragments are size-fractionated according tostandard procedures. EcoRI linkers are ligated to the DNA and thefragments then ligated into the vector Lambda ZapII that have been cutwith EcoRI, the library packaged by standard procedures, and E.coliinfected with the packaged library. The library is amplified by standardprocedures.

Example 2 tktA Characterization

[0132] We have used chemical mutagenesis to isolatetemperature-sensitive (ts) mutants in an attempt to identify essentialP. aeruginosa gene products. Over 100 mutants, which show ts growth oncomplex medium at 44° C., have been isolated. A genomic librarycontaining 5 to 6 kb DNA fragments of wild type P. aeruginosa wasconstructed to complement these ts mutants. Nucleotide sequence analysisof plasmids complementing the ts mutants revealed many known essentialgenes as well as genes with unknown functions. One of the ts mutants,ts-92, was shown to have mutations in the tktA gene encodingtransketolase A. Mutant ts-92 contains a G→A transition mutation atnucleotide position 611 in Table 1 [SEQ ID NO:1], which caused an aminoacid substitution resulting in the change of arginine at position 204 inTable 1 [SEQ ID NO:2] to histidine in the TktA ORF. The resultsdemonstrated that the tktA gene product is essential for cell growth invitro and could be used as an antimicrobial target.

1 2 1 1998 DNA Pseudomonas aeruginosa 1 atgcccagcc gtcgtgagcg agccaatgccatccgtgcac tgagcatgga tgccgtgcag 60 aaagccaaca gcggccaccc gggcgccccgatgggcatgg ccgatatcgc cgaggtcctc 120 tggcgcgact acatgcagca caacccgagcaacccgcagt gggccaaccg cgaccgcttc 180 gtgctgtcca acggccacgg ctcgatgctgatctactccc tgctgcacct caccgggtac 240 gacctcggca tcgaggacct gaagaacttccgccagctca actcgcgcac cccgggccac 300 ccggagtacg gctacaccgc cggcgtcgagaccaccaccg gtccgctcgg ccagggcatc 360 gccaatgcgg tgggcatggc gctggcggagaaggtcctgg ccgcccagtt caaccgcgac 420 ggccacgcgg tggtcgacca ctacacctacgccttcctcg gcgacggctg catgatggaa 480 ggcatttccc atgaggtcgc ctcgctggccggcaccctgc gcctgaacaa gctgatcgcc 540 ttctacgacg acaacggcat ttccatcgacggcgaggtcc acggctggtt caccgacgac 600 accccgaagc gcttcgaggc ctatggctggcaagtgatcc gcaacgtcga cgggcatgac 660 gccgacgaga tcaagaccgc catcgataccgcgcgcaaga gcgaccagcc gaccctgatc 720 tgctgcaaga ccgtgatcgg tttcggctcgccgaacaagc agggcaagga agagtgccac 780 ggcgcgccgc tgggcgccga cgagatcgccgcgacccgcg ccgcgctggg ctgggagcac 840 gctccgttcg agatcccggc gcagatctacgccgagtggg acgccaagga aaccggcgcc 900 gcccaggaag ccgagtggaa caagcgtttcgccgcctacc aggctgccca tccggaactg 960 gccgccgaat tgctgcgccg cctgaagggcgagctgccgg ccgacttcgc cgagaaggcc 1020 gcggcctacg tcgccgatgt tgccaacaagggtgagacca tcgccagccg caaggccagc 1080 cagaacgcgc tgaacgcctt cggcccgctgctgccggagc tgctcggcgg ttccgccgac 1140 ctggccggct ccaacctgac cttgtggaagggctgcaagg gcgtcagcgc cgacgacgcc 1200 gccggcaact acgtgttcta cggcgtgcgcgaattcggca tgagcgcgat catgaatggc 1260 gtcgccctgc acggcggttt cattccctacggtgcgacct tcctgatctt catggaatac 1320 gcgcgcaacg ccgtgcgcat gtccgcactgatgaagcagc gcgtgctcta cgtgttcacc 1380 cacgactcca tcggcctcgg cgaggacggcccgacccacc agccgatcga acaactggcc 1440 agcctgcgcc tgaccccgaa cctggacacctggcgcccgg ccgacgcggt cgagtcggcg 1500 gtggcctgga agcatgccat cgagcgcgccgacggtccgt ccgcgctgat cttctcccgc 1560 cagaacctgc cgcaccaggc gcgcgacgtcgcccaggtgg ccgacatcgc ccgcggcggc 1620 tacgtgctga aggactgcga aggcgagccggaactgatcc tgatcgccac cggttcggaa 1680 gtcggcctgg ccgtgcaggc ctacgacaagctcagcgagc agggccgcaa ggtccgcgtg 1740 gtatcgatgc catgcaccag cgtctacgagcagcaggacg agtcctacaa gcagtccgtg 1800 ctgccggtgg aagtcggcgc gcgcatcgccatcgaggccg cccatgccga ctactggtac 1860 aagtacgtcg gtctcgacgg gcgcatcatcggcatgacca gcttcggcga gtcggcgccg 1920 gccccggcgc tgttcgagca cttcggcttcaccctggaca acgtcctggc ggtgggcgag 1980 gagctgctgg aagactga 1998 2 665 PRTPseudomonas aeruginosa 2 Met Pro Ser Arg Arg Glu Arg Ala Asn Ala Ile ArgAla Leu Ser Met 1 5 10 15 Asp Ala Val Gln Lys Ala Asn Ser Gly His ProGly Ala Pro Met Gly 20 25 30 Met Ala Asp Ile Ala Glu Val Leu Trp Arg AspTyr Met Gln His Asn 35 40 45 Pro Ser Asn Pro Gln Trp Ala Asn Arg Asp ArgPhe Val Leu Ser Asn 50 55 60 Gly His Gly Ser Met Leu Ile Tyr Ser Leu LeuHis Leu Thr Gly Tyr 65 70 75 80 Asp Leu Gly Ile Glu Asp Leu Lys Asn PheArg Gln Leu Asn Ser Arg 85 90 95 Thr Pro Gly His Pro Glu Tyr Gly Tyr ThrAla Gly Val Glu Thr Thr 100 105 110 Thr Gly Pro Leu Gly Gln Gly Ile AlaAsn Ala Val Gly Met Ala Leu 115 120 125 Ala Glu Lys Val Leu Ala Ala GlnPhe Asn Arg Asp Gly His Ala Val 130 135 140 Val Asp His Tyr Thr Tyr AlaPhe Leu Gly Asp Gly Cys Met Met Glu 145 150 155 160 Gly Ile Ser His GluVal Ala Ser Leu Ala Gly Thr Leu Arg Leu Asn 165 170 175 Lys Leu Ile AlaPhe Tyr Asp Asp Asn Gly Ile Ser Ile Asp Gly Glu 180 185 190 Val His GlyTrp Phe Thr Asp Asp Thr Pro Lys Arg Phe Glu Ala Tyr 195 200 205 Gly TrpGln Val Ile Arg Asn Val Asp Gly His Asp Ala Asp Glu Ile 210 215 220 LysThr Ala Ile Asp Thr Ala Arg Lys Ser Asp Gln Pro Thr Leu Ile 225 230 235240 Cys Cys Lys Thr Val Ile Gly Phe Gly Ser Pro Asn Lys Gln Gly Lys 245250 255 Glu Glu Cys His Gly Ala Pro Leu Gly Ala Asp Glu Ile Ala Ala Thr260 265 270 Arg Ala Ala Leu Gly Trp Glu His Ala Pro Phe Glu Ile Pro AlaGln 275 280 285 Ile Tyr Ala Glu Trp Asp Ala Lys Glu Thr Gly Ala Ala GlnGlu Ala 290 295 300 Glu Trp Asn Lys Arg Phe Ala Ala Tyr Gln Ala Ala HisPro Glu Leu 305 310 315 320 Ala Ala Glu Leu Leu Arg Arg Leu Lys Gly GluLeu Pro Ala Asp Phe 325 330 335 Ala Glu Lys Ala Ala Ala Tyr Val Ala AspVal Ala Asn Lys Gly Glu 340 345 350 Thr Ile Ala Ser Arg Lys Ala Ser GlnAsn Ala Leu Asn Ala Phe Gly 355 360 365 Pro Leu Leu Pro Glu Leu Leu GlyGly Ser Ala Asp Leu Ala Gly Ser 370 375 380 Asn Leu Thr Leu Trp Lys GlyCys Lys Gly Val Ser Ala Asp Asp Ala 385 390 395 400 Ala Gly Asn Tyr ValPhe Tyr Gly Val Arg Glu Phe Gly Met Ser Ala 405 410 415 Ile Met Asn GlyVal Ala Leu His Gly Gly Phe Ile Pro Tyr Gly Ala 420 425 430 Thr Phe LeuIle Phe Met Glu Tyr Ala Arg Asn Ala Val Arg Met Ser 435 440 445 Ala LeuMet Lys Gln Arg Val Leu Tyr Val Phe Thr His Asp Ser Ile 450 455 460 GlyLeu Gly Glu Asp Gly Pro Thr His Gln Pro Ile Glu Gln Leu Ala 465 470 475480 Ser Leu Arg Leu Thr Pro Asn Leu Asp Thr Trp Arg Pro Ala Asp Ala 485490 495 Val Glu Ser Ala Val Ala Trp Lys His Ala Ile Glu Arg Ala Asp Gly500 505 510 Pro Ser Ala Leu Ile Phe Ser Arg Gln Asn Leu Pro His Gln AlaArg 515 520 525 Asp Val Ala Gln Val Ala Asp Ile Ala Arg Gly Gly Tyr ValLeu Lys 530 535 540 Asp Cys Glu Gly Glu Pro Glu Leu Ile Leu Ile Ala ThrGly Ser Glu 545 550 555 560 Val Gly Leu Ala Val Gln Ala Tyr Asp Lys LeuSer Glu Gln Gly Arg 565 570 575 Lys Val Arg Val Val Ser Met Pro Cys ThrSer Val Tyr Glu Gln Gln 580 585 590 Asp Glu Ser Tyr Lys Gln Ser Val LeuPro Val Glu Val Gly Ala Arg 595 600 605 Ile Ala Ile Glu Ala Ala His AlaAsp Tyr Trp Tyr Lys Tyr Val Gly 610 615 620 Leu Asp Gly Arg Ile Ile GlyMet Thr Ser Phe Gly Glu Ser Ala Pro 625 630 635 640 Ala Pro Ala Leu PheGlu His Phe Gly Phe Thr Leu Asp Asn Val Leu 645 650 655 Ala Val Gly GluGlu Leu Leu Glu Asp 660 665

What is claimed is:
 1. An isolated polypeptide selected from the groupconsisting of: (i) an isolated polypeptide comprising an amino acidhaving at least 95% identity to the amino acid sequence of SEQ ID NO:2over the entire length of SEQ ID NO:2; (ii) an isolated polypeptidecomprising the amino acid sequence of SEQ ID NO:2, (iii) an isolatedpolypeptide that is the amino acid sequence of SEQ ID NO:2, and (iv) apolypeptide that is encoded by a recombinant polynucleotide comprisingthe polyncleotide sequence of SEQ ID NO:1.
 2. An isolated polynucleotideselected from the group consisting of: (i) an isolated polynucleotidecomprising a polynucleotide sequence encoding a polypeptide that has atleast 95% identity to the amino acid sequence of SEQ ID NO:2, over theentire length of SEQ ID NO:2; (ii) an isolated polynucleotide comprisinga polynucleotide sequence that has at least 95% identity over its entirelength to a polynucleotide sequence encoding the polypeptide of SEQ IDNO:2; (iii) an isolated polynucleotide comprising a nucleotide sequencethat has at least 95% identity to that of SEQ ID NO:1 over the entirelength of SEQ ID NO:1; (iv) an isolated polynucleotide comprising anucleotide sequence encoding the polypeptide of SEQ ID NO:2; (v) anisolated polynucleotide that is the polynucleotide of SEQ ID NO:1; (vi)an isolated polynucleotide of at least 30 nucleotides in lengthobtainable by screening an appropriate library under stringenthybridization conditions with a probe having the sequence of SEQ ID NO:1or a fragment thereof of of at least 30 nucleotides in length; (vii) anisolated polynucleotide encoding a mature polypeptide expressed by thetktA gene comprised in the Pseudomonas aeruginosa; and (viii) apolynucleotide sequence complementary to said isolated polynucleotide of(i), (ii), (iii), (iv), (v), (vi) or (vii).
 3. A method for thetreatment of an individual: (i) in need of enhanced activity orexpression of or immunological response to the polypeptide of claim 1comprising the step of: administering to the individual atherapeutically effective amount of an antagonist to said polypeptide;or (ii) having need to inhibit activity or expression of the polypeptideof claim 1 comprising: (a) administering to the individual atherapeutically effective amount of an antagonist to said polypeptide;or (b) administering to the individual a nucleic acid molecule thatinhibits the expression of a polynucleotide sequence encoding saidpolypeptide; (c) administering to the individual a therapeuticallyeffective amount of a polypeptide that competes with said polypeptidefor its ligand, substrate, or receptor; or (d) administering to theindividual an amount of a polypeptide that induces an immunologicalresponse to said polypeptide in said individual.
 4. A process fordiagnosing or prognosing a disease or a susceptibility to a disease inan individual related to expression or activity of the polypeptide ofclaim 1 in an individual comprising the step of: (a) determining thepresence or absence of a mutation in the nucleotide sequence encodingsaid polypeptide in an organism in said individual; or (b) analyzing forthe presence or amount of said polypeptide expression in a samplederived from said individual.
 5. A process for producing a polypeptideselected from the group consisting of: (i) an isolated polypeptidecomprising an amino acid sequence selected from the group having atleast 95% identity to the amino acid sequence of SEQ ID NO:2 over theentire length of SEQ ID NO:2; (ii) an isolated polypeptide comprisingthe amino acid sequence of SEQ ID NO:2; (iii) an isolated polypeptidethat is the amino acid sequence of SEQ ID NO:2, and (iv) a polypeptidethat is encoded by a recombinant polynucleotide comprising thepolynucleotide sequence of SEQ ID NO:1, comprising the step of culturinga host cell under conditions sufficient for the production of thepolypeptide.
 6. A process for producing a host cell comprising anexpression system or a membrane thereof expressing a polypeptideselected from the group consisting of: (i) an isolated polypeptidecomprising an amino acid sequence selected from the group having atleast 95% identity to the amino acid sequence of SEQ ID NO:2 over theentire length of SEQ ID NO:2; (ii) an isolated polypeptide comprisingthe amino acid sequence of SEQ ID NO:2; (iii) an isolated polypeptidethat is the amino acid sequence of SEQ ID NO:2, and (iv) a polypeptidethat is encoded by a recombinant polynucleotide comprising thepolynucleotide sequence of SEQ ID NO:1, said process comprising the stepof transforming or transfecting a cell with an expression systemcomprising a polynucleotide capable of producing said polypeptide of(i), (ii), (iii) or (iv) when said expression system is present in acompatible host cell such the host cell, under appropriate cultureconditions, produces said polypeptide of (i), (ii), (iii) or (iv).
 7. Ahost cell or a membrane expressing a polypeptide selected from the groupconsisting of: (i) an isolated polypeptide comprising an amino acidsequence selected from the group having at least 95% identity to theamino acid sequence of SEQ ID NO:2 over the entire length of SEQ IDNO:2; (ii) an isolated polypeptide comprising the amino acid sequence ofSEQ ID NO:2; (iii) an isolated polypeptide that is the amino acidsequence of SEQ ID NO:2, and (iv) a polypeptide that is encoded by arecombinant polynucleotide comprising the polynucleotide sequence of SEQID NO:1.
 8. An antibody immunospecific for the polypeptide of claim 1.9. A method for screening to identify compounds that agonize or thatinhibit the function of the polypeptide of claim 1 that comprises amethod selected from the group consisting of: (a) measuring the bindingof a candidate compound to the polypeptide (or to the cells or membranesbearing the polypeptide) or a fusion protein thereof by means of a labeldirectly or indirectly associated with the candidate compound; (b)measuring the binding of a candidate compound to the polypeptide (or tothe cells or membranes bearing the polypeptide) or a fusion proteinthereof in the presence of a labeled competitor; (c) testing whether thecandidate compound results in a signal generated by activation orinhibition of the polypeptide, using detection systems appropriate tothe cells or cell membranes bearing the polypeptide; (d) mixing acandidate compound with a solution comprising a polypeptide of claim 1,to form a mixture, measuring activity of the polypeptide in the mixture,and comparing the activity of the mixture to a standard; or (e)detecting the effect of a candidate compound on the production of mRNAencoding said polypeptide and said polypeptide in cells, using forinstance, an ELISA assay.
 10. An agonist or antagonist to thepolypeptide of claim 1.